The nucleotide sequence of replication and maintenance functions encoded by plasmid PSCl0l

Churchward, Gordon; Linder, Patrick; Caro, Lucien

doi:10.1093/nar/11.16.5645

Cited by 91 publications

(49 citation statements)

References 47 publications

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“…The coordinates used throughout the text are those of the origin region of pSC101, as defined in Churchward et al (10). We (10).…”

Section: Methodsmentioning

confidence: 99%

“…USA 89:8923-8927, 1992 The functions and sites required for the replication of plasmid pSC101 (11) in Escherichia coli lie within a 2.2-kb HincII-RsaI fragment of the plasmid (Fig. 1) (2,10,23,25,26,28,37,41). The region contains three directly repeated sequences, RS1, RS2, and RS3 and two inversely repeated sequences, IR1 and IR2, partially homologous to the direct repeats.…”

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confidence: 99%

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In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101

Xia

Manen

et al. 1993

J Bacteriol

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The RepA replication protein of plasmid pSC101 binds as a monomer to three repeated sequences (RS1, RS2, and RS3) in the replication origin of the plasmid to initiate duplication and binds as a dimer to two inversely repeated sequences (IR1 and ER2) in its promoter region (D. Manen, L. C. Upegui-Gonzalez, and L. Caro, Proc. Natl. Acad. Sci. USA 89:8923-8927, 1992 The functions and sites required for the replication of plasmid pSC101 (11) in Escherichia coli lie within a 2.2-kb HincII-RsaI fragment of the plasmid (Fig. 1) (2,10,23,25,26,28,37,41). The region contains three directly repeated sequences, RS1, RS2, and RS3 and two inversely repeated sequences, IR1 and IR2, partially homologous to the direct repeats. It encodes a 37-kDa, 316-amino-acid-long initiator protein, RepA, which acts at at least two levels: positively, it initiates plasmid replication by binding to the repeated sequences RS1, RS2, and RS3 in the on locus; negatively, it autoregulates its transcription by binding to the palindromic sequences IR1 and IR2 that overlap its promoter (24,36,38). In addition, pSC101 replication requires several E. coli proteins, in particular IHF (17) and DnaA (14,19). Both bind to specific sites in the origin region (Fig. 1). Sequences rich in AT bracket the IHF site. The DNA of the on segment bends around the IHF binding site, and the curvature is enhanced by the binding of IHF (34). Interactions between DnaA, IHF, and RepA in their binding reactions to the origin of pSC101 have been demonstrated (33).Previous work in our laboratory has shown that a point mutation in the repA gene, changing amino acid 93 from glutamic acid to lysine in the protein, results in a four-to fivefold increase in plasmid copy number and a 1.7-fold increase in the amount of protein produced, pointing to a * Corresponding author. dual effect on replication of the plasmid and on autoregulation of the protein (40). Band shift assays performed with crude extracts indicated that the mutant protein RepA(cop) was bound more efficiently than the wild-type protein to repeated sequences in the origin. To verify that the changes were produced by the mutation in the protein rather than by a modification of the origin structure due to the mutation, we have studied its effects in vivo when provided in trans. To further elucidate the properties of the mutant and of the wild-type proteins, we have purified them and studied their binding to directly repeated sequences in the origin and to palindromic sequences in the promoter region. We have confirmed that the purified protein binds the repeated sequences more efficiently and the palindromic sequences less efficiently than does the wild-type protein.

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“…The coordinates used throughout the text are those of the origin region of pSC101, as defined in Churchward et al (10). We (10).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101

Xia

Manen

et al. 1993

J Bacteriol

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“…515-294-1525, Fax: 515-294-1401 gregory@iastate.edu. (Armstrong et al, 1984) through binding repeated sequences known as iterons at the ori (Chattoraj, 2000;Churchward et al, 1983;Vocke and Bastia, 1983). RepA also autoregulates its own expression by binding as a dimer to inverted repeats near the repA promoter (Linder et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

New pSC101-derivative cloning vectors with elevated copy numbers

Peterson

Phillips

2008

Plasmid

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Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (∼240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the E. coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.

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“…The AT-rich region in the pSC101 origin is similar to that of oriC and contains two copies of the same 13-mer sequence (8,52). However, although DnaA also binds to the pSC101 origin (45), the orientation of the strong DnaA binding site with respect to the 13-mer sequences is different from that found in oriC (45).…”

mentioning

confidence: 95%

“…1) (24,52,58). The most striking features of this region are an AT-rich tract, which contains a binding site for IHF, and three tandemly repeated sequences, denoted RS1 to RS3 (26), which bind Rep protein (8,46,50,52,53). Immediately adjacent to ori lies the promoter region of the rep gene (25).…”

mentioning

confidence: 99%

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein

1992

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The nucleotide sequence of replication and maintenance functions encoded by plasmid PSCl0l

Cited by 91 publications

References 47 publications

In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101

In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101

New pSC101-derivative cloning vectors with elevated copy numbers

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein

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